1. Field of the Invention
This invention relates to detection assays useful in providing quantitative measurements of dengue virus as well as providing qualitative detection of any dengue serotype virus in research samples.
2. Description of the Prior Art
Dengue viruses are a major public health concern with serious medical and economic consequences and are currently considered the most important arthropod disease affecting humans in terms of morbidity and mortality. (1,2) Dengue fever is endemic in most tropical and subtropical areas worldwide and several hundred thousand dengue hemorrhagic fever cases are reported to occur annually. (3) Due to the vast expansion of air travelling, new dengue virus strains may introduced into a susceptible population in the tropics. Also tourists with dengue fever are now frequently seen in areas where dengue fever is not endemic and where physicians are not familiar with the disease. As symptoms of dengue fever are usually non-specific, a reliable diagnosis is difficult to obtain unless virological techniques are included. (3)
Both dengue virus-specific immunoglobulin G (IgG) and IgM antibodies are usually found in the sera from patients with acute primary infections, while the IgM response may be low or sometimes even absent in secondary dengue fever. (3) However, a strong antibody cross-reactivity exists among the flavivirus family. Therefore, the antibody response may be difficult to interpret with regard to an acute dengue fever, if other flavivirus infections cannot be excluded by clinical laboratory, or epidemiological means. (3)
Previous methods of quantitating dengue viremia involved the isolation of virus from samples using tissue culture, IFA (immunofluorescent antibody), and plaque titer methods. These classical methods are considered the gold standard; however, these methods are tedious, slow, and often difficult to standardize, and require specialized expertise. The total turn-around time is often two to three weeks and the isolation rates and sensitivity are low. Laue et al., reports the detection of dengue virus RNA by reverse transcriptase PCR (RT-PCR) in human serum or plasma samples is highly indicative of acute dengue fever. (3) Moreover this method is able to identify the dengue virus serotype by demonstrating defined sequence homologies in the viral genomic RNA. Unfortunately, the technique of RT-PCR is handicapped both by time-consuming nested amplification protocols and by false positive reactions which may in part be due to the contamination of dengue virus DNA in the laboratory. (3)
Laue et al sought to overcome this problem by applying a fully automated amplification protocol which sensitively detects the four serotypes but avoids DNA contamination. The protocol uses the TaqMan priniciple by monitoring a fluorescent signal which tracks the increase in dengue virus-specific DNA during amplification in tightly sealed test tubes. This protocol was viewed as being a simple, highly specific and sensitive test since the test tubes no longer needed to be opened, as in previous methods, to quantitate the PCR product. (3)
Figuerido et al. teach a simplified RT-PCR for identification of dengue virus types 1 and 2 in a single reaction vessel, carried out in a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, dengue virus consensus primers having maximum sequence similarity to the four serotypes was used which amplified a 511 base pair sequence. The reaction mixture contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1 U of thermostable Taq DNA polymerase. The mixture was incubated followed by 2-step PCR amplification with slow temperature increment. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. This technique was found to be less laborious, faster, with reduced risk of contamination.